Ginseng (Panax ginseng C. A meyer) is one of the most popular medicinal herbs widely used for improving health. The root of ginseng was consumed as a herbal tea in a traditional medicine, and currently it has been included in a variety of products including candy, instant tea, and tonic drink. Ginsenosides which are glycosylated triterpenes contained in ginseng have positive effects on health. In particular, ginsenosides have been known to have various pharmacological effects such as immune system enhancement and revitalization of body functions. Also more than 40 different ginsenosides have been identified in the root of ginseng. However, since mass production of each of the ginsenosides is hard, it remains as a major obstacle for investigating efficacy of certain ginsenoside, for example, its therapeutic effects on specific diseases.
Ginsenosides are glycosylated dammarene-type tetracyclic triterpenes, and can be classified into three different groups based on their aglycone structure: Protopanaxadiol (PPD)-type ginsenosides, Protopanaxatriol (PPT)-type ginsenosides, and Oleanolic acid-type ginsenosides. These three groups can be further classified based on the position and number of sugar moieties (aglycones) attached to the C-3, C-6, and C-20 positions of the rings by a glycosidic bond in the chemical structure. PPD and PPT also possess different hydroxylation patterns. PPD possesses —OH groups at the C-3, C-12, and C-20 positions, whereas PPT possesses —OH groups at the C-3, C-6, C-12, and C-20 positions. PPD and PPT can be glycosylated with glucose and/or other types of sugars to be converted into various ginsenosides. The representative PPD-type ginsenosides include ginsenoside Rh2, ginsenoside Rg3, Compound K (C-K), ginsenoside F2, and ginsenoside Rd. And the representative PPT-type ginsenosides include ginsenoside F1, Rg1, Re, Rh1, and Rg2.
The biosynthetic pathway of ginsenosides is only partially identified. The ginsenoside biosynthesis is known to share the biosynthetic pathways with other triterpenes until oxidosqualene is synthesized by a series of condensation reactions of isopentenyl diphosphate and DMADP (dimethylallyl diphosphate) by the action of IPP isomerase (IPI), GPP synthase (GPS), FPP synthase (FPS), squalene synthase (SS) and squalene epoxidase (SE) (Ajikumar et al. Science, 330, 70-74. 2010; Ro et al. Nature, 440, 940-943. 2006; Sun et al. BMC genomics, 11, 262, 2010). Oxidosqualene is cyclized into dammarenediol-II by DS (dammarenediol-II synthase) which is a triterpene cyclase. Dammarenediol-II has hydroxyl groups at the C-3 and C-20 positions, and is converted into PPD by hydroxylation of the C-12 position by a p450 enzyme, PPDS (protopanaxadiol synthase). PPDS can be also converted into PPT by hydroxylation at the C-6 position by another p450 enzyme, PPTS (protopanaxatriol synthase). PPD can be converted into PPD-type ginsenoside by glycosylation at the C-3 and/or C-20 position(s), and PPT can be converted into PPT-type ginsenoside by glycosylation at the C-6 and/or C-20 position(s). UDP (Uridine diphosphate)-glycosyltransferase (UGT) is considered to be involved in synthetic pathways of various ginsenosides by formation of O-, β1,2-, or β1,6-glycosidic linkage. DS, PPDS and PPTS have been reported as the enzymes involved in ginsenoside biosynthesis, but it has not been identified whether UGT is involved in the biosynthesis of ginsenosides.
UDP-glycosyltransferase is an enzyme that catalyzes the transfer of a sugar moiety from UDP-sugar to a wide range of metabolites such as hormones and secondary metabolites. Generally, UGT acts in the final step of biosynthetic pathway in order to increase solubility, stability, storage, bioactivity, or biological availability of metabolites. As recognized by a remarkable diversity of metabolites in plants, the genome of a plant possesses hundreds of different UGTs. For example, a plant model, Arabidopsis thaliana contains 107 UGTs that belong to 14 different groups (Group A to Group N) based on the amino acid sequence. Different UGTs show substrate specificity towards both sugar donor and sugar acceptor. For example, UGT78D2 transfers glucose from UDP-glucose to the C-3 position of flavonol (kaempferol, quercetin) and anthocyanin (cyanidin) in order to produce flavonol 3-O-glucosides and cyanidin 3-O-glucoside, respectively. It seems that such glycosylation is essential for in vivo stability and storage of the compound. On the other hand, UGT89C1 transfers rhanmnose from UDP-rhanmnose to the C-7 position of flavonol-3-O-glucosides in order to produce flavonol-3-O-glucoside-7-O-rhamnoside. Likewise, UGT89C1 does not utilize UDP-glucose and anthocyanin-3-O-glucoside as a substrate. And it has different specificity towards UDP-sugar and acceptor from that of UGT78D2. Therefore, there is a need to investigate the substrate specificity for different types of UGTs.